Prolidase is a unique enzyme responsible for the hydrolysis of endogenous and exogenous X-Pro dipeptides during protein absorption and metabolism. It also participates in the collagen metabolism and endogenous Pro recycling. Prolidase deficiency, an autosomal recessive genetic disorder, is associated with severe development disorders in children including mental retardation. Despite this apparent association, the role played by prolidase in the pathogenesis of this genetic disorder is unclear. Therefore, additional studies are necessary to determine the pathogenesis of this disease in order to develop effective treatment plans. This study aims to determine factors associated with the regulation of different prolidase isozymes in the intestine to further elucidate the functions of prolidase. The rationale is that better characterization of each isozyme's physiological functions will lead to better understanding of their roles in the pathogenesis. The short term goals of the proposed research ar to purify rat intestinal prolidase, and to determine polymorphism and site-specific regulation of rat intestinal prolidase by diet and chemicals. Intestinal prolidase is selected because intestine is the first organ where dietary X- Pro is hydrolyzed. The specific aims are to: (1) purify a large quantities (50- 100 ug) of prolidase I, and characterize this isozyme by determining its kinetic characteristics (e.g., km), substrate specificities, pH-activity profile, and inhibitor effects. Purify or at least partially purify prolidase II in sufficient quantity to perform preliminary characterization similar to those performed for prolidase I; (2) determine prolidase polymorphism at different regions of the intestine; and (3) study the regulation of prolidase isozymes and determine if one isozyme is more susceptible to the effects of regulation than the other. The long term goals of this study are to determine the physiological functions of this enzyme and to develop viable treatment strategy or monitoring tool for patients suffering from the severe symptoms related to prolidase deficiency, or other diseases associated with elevated level of plasma prolidase.